1.Screening Of High-Affinity Binding Peptide Of DEP Domain Of Dvl Protein By Phage Display 2.Role Of DEP Domain In Membrane Translocation Of Dvl-1

Wnt signaling plays the important roles in embryogenesis and tumorgenesis. Although the mechanism about how Wnts transduct their signaling from receptor frizzled (Fz) to cytosol has not been well understood, dishevelled (Dvl) protein is believed as the focus of this process. The previous research found that the DEP domain of Dvl alone may interact with an upstream partner(s), subsequently the interaction blocks the activity of upstream signalling of Dvl, while a KM mutant (Lys434 mutated to Met) of Dvl does not possess this function.In the first part of this thesis, we used DEP domain of Dvl as bait and its functional mutation K434M as a control to screen high affinity binding peptide in bacteria phage display system. After four rounds of screening, we got a series of positive clones containing certain peptides, which can specially bind to DEP domain. After sequence analysis, an extremely rich peptide was found:TFWDDGWLLLPR(P12). This peptide is mainly composed of Trp and other acidic amino acid, which obviously match to the surface activity site of DEP reported previously. The low affinity between P12 and DEP-KM may be caused by the loss of a basic amino acid in this surface activity site. Further more, GST pull-down assay showed that GST-fused P12 tightly binds to DEP, but the affinity between GST-P12 and DEP-KM is much lower. We also found that P12 can inhibit the transcriptional activity induced by Wnt3a, but not Dvl-1 or a constitutive form of (-catenin - (N-(-catenin in LEF reporter gene assay. In the second part, we characterized the function of three domains (DIX, PDZ and DEP) of Dvl-1 in canonical Wnt signal transduction and Dvl-1 membrane translocation. Both DIX and DEP domain was sufficient to block Wnt-3a-induced LEF-1 transcriptional activity and free cytosol (-catenin accumulation; whereas PDZ domain and a KM mutant form of DEP domain (DEP-KM) had no effect on canonical Wnt signaling. In addition, when cotransfected with Fz-7, DEP domain, but not DIX, PDZ or DEP-KM, was translocated and co-localized with Fz-7 to the cytosol plasma as similar as Dvl-1. Furthermore, DEP domain, but not DIX, PDZ or DEP-KM, can block Fz-7-induced membrane translocation of Dvl-1 via a possible competitive mechanism. These results strongly suggest that DEP domain is responsible for the membrane translocation of Dvl-1 protein upon Wnt signal stimulation. ...